Figure 5.
Figure 5. A conserved cluster of three acidic residues is required for full-length VE-cadherin endocytosis. (A and B) Wild-type (WT) or mutant VE-cadherin–RFP was expressed in COS-7 cells, and endocytosis was measured using a fluorescence-based internalization assay with an internalization period of 30 min. Antibody labeling and low pH wash was used to identify internalized (intern.) cadherin (A, left column), and the RFP tag was used to identify total cadherin (A, right column). Means ± SEM (n = 20–24 cells per group); ***, P < 0.001 compared with wild type and GGG mutation; *, P < 0.05 compared with wild type. (C) Time-lapse images of wild-type and DEE mutant VE-cadherin (VE-cad)–RFP expressed in primary human microvascular endothelial cells. Time is shown in minutes and seconds. The arrowheads highlight an endocytic event. See also Videos 1 and 2. Bars: (A) 20 µm; (C) 10 µm.

A conserved cluster of three acidic residues is required for full-length VE-cadherin endocytosis. (A and B) Wild-type (WT) or mutant VE-cadherin–RFP was expressed in COS-7 cells, and endocytosis was measured using a fluorescence-based internalization assay with an internalization period of 30 min. Antibody labeling and low pH wash was used to identify internalized (intern.) cadherin (A, left column), and the RFP tag was used to identify total cadherin (A, right column). Means ± SEM (n = 20–24 cells per group); ***, P < 0.001 compared with wild type and GGG mutation; *, P < 0.05 compared with wild type. (C) Time-lapse images of wild-type and DEE mutant VE-cadherin (VE-cad)–RFP expressed in primary human microvascular endothelial cells. Time is shown in minutes and seconds. The arrowheads highlight an endocytic event. See also Videos 1 and 2. Bars: (A) 20 µm; (C) 10 µm.

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